Lipids have been shown to have important roles in an intracellular signal transmission system. In particular, researches on signal transmission by phosphates of phosphatidylinositol are actively conducted.
Phosphatidylinositol (PI) is a substance that inositol is bound as a phosphate ester to a remaining hydroxyl group of diacylglycerol. Phosphatidylinositol (PI) is one kind of phospholipids that is found on membranes of cells, however it occupies less than 10% of the total phospholipids. Phosphatidylinositol (PI) plays two major roles. First, it remains on the membrane and serves as a scaffold for a variety of phosphorylation reactions for a phosphorylated enzyme and the like. Second, it serves to release phosphorylated inositol as a second messenger upon hydrolysis of the phosphorylated inositol moiety.
Hydroxyl groups in the inositol moiety of PI may further be phosphorylated, and inositol phospholipids in which 3-position, 4-position or 5-position or two or more of these positions is/are phosphorylated are also known. In particular, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) in which 4-position and 5-position are phosphorylated not only forms an intermediate of phosphatidylinositol 3,4,5-triphosphate (PI(3,4,5)P3) that plays a role in vesicular transport and signal transmission to a nucleus, but also plays an important role by itself.
PI(4,5)P2 is able to bind to a variety of actin binding proteins, and serves to keep and change the shape of a cell by allowing rearrangement of actin in the cell. Another important role of the PI(4,5)P2 is that it is hydrolyzed by phospholipase C (PLC) into inositol-1,4,5-triphosphate (IP3) and diacylglycerol (DAG) which are then released as second messengers. The released inositol-1,4,5-triphosphate (IP3) binds to an IP3 receptor (IP3R) residing on the surface of an endoplasmic reticulum of a cell and release Ca2+ stored in endoplasmic reticula into the cytoplasm, thereby activating a variety of Ca2+ dependent enzymes. DAG also activates C kinase.
Upon binding of substances such as hormones, cytokines and neurotransmitters on receptors of a cell, production of inositol-1,4,5-triphosphate (IP3) is induced in the cell, and the IP3 binds to inositol-1,4,5-triphosphate (IP3) receptor (IP3R) residing on the surface of endoplasmic reticula of the cell. The IP3R converts IP3 signaling into Ca2+ signaling, and thus plays crucial roles in a wide range of cellular functions such as embryonic development, differentiation of cells, proliferation, phagocytosis, granule secretion, motion and actions in nerve system, and it is especially important for the IP3R to normally function in cells in order to keep the homeostasis of cells. Thus, IP3R is not only an important protein involved in signal transmission of cells, but also an important protein capable of regulating a variety of functions of cells. Therefore, modulation of the function of IP3R makes it possible to adjust a variety of actions in cells.
As the significance of IP3 or IP3R in a cell becomes clear, researches on these proteins are conducted more actively. As such, a patent application relating to a monoclonal antibody against IP3R (See prior art document 1, Japanese Patent Application Laid-open No. 8-134099), a patent application relating to a method of competition for IP3 (See prior art document 2, Japanese Translation of PCT International Application No. 8-502068) and a patent application relating to polypeptides that bind to IP3R with high affinity (see prior art document 3, Japanese Patent Application Laid-open No. 2000-135095) have been filed.
So far, molecular cloning studies have revealed that the IP3R family in mammalian consists of at least three subtypes. (see prior art documents 4 to 6, Furuichi, T., Yoshikawa, S., et al., (1989), Nature, 342, 32-38; Blondel, O., Takeda, J., et al., (1993), J. Biol. Chem., 268, 11356-11363; Yamamoto-Hino, M., Sugiyama, T., et al., (1994), Receptors Channels, 2, 9-22). Structural and functional studies have shown that IP3Rs are composed of three domains: ligand binding, modulatory, and channel (see prior art document 7, Furuichi, T., Kohda, K., et al., (1994), Curr. Opin. Neurobiol., 4, 294-303).
The binding domain which is N-terminal stretch of approximate 600 amino acids in IP3R is responsible for binding of IP3. Mutational analysis showed three amino acid residues in this region, for example, Arg-265, Lys-508 and Arg-511 in mouse IP3R type 1 are critical for IP3 binding, and Arg-658 is determinant for binding specificity to various inositol phosphates.
The channel domain has six membrane spanning segments, which are clustered near the C-terminus of IP3R and correspond to Ca2+ channel. With respect of first to fourth membrane spanning domains, high homology is maintained in the IP3R family.
The modulatory domain resides between the N-terminal binding domain and the C-terminal channel domain, and the modulatory domain contains binding sites for various modulators such as Ca2+, Ca2+-calmodulin, FK506 binding protein 12K, ATP, and sites for phosphorylation by cAMP-dependent protein kinase, cGMP-dependent protein kinase and protein kinase C. This modulatory domain may transduce IP3-binding to channel opening. IP3-induced Ca2+ release maybe regulated by various modifications in the modulatory domain. Interestingly, homology of this region among the IP3R family is low in comparison with other regions and results in different modifications between subtypes, which suggests that each type of IP3R/Ca2+ channel function may be differently modulated to produce unique channel properties.
Since the function of IP3R is finely adjusted by substances that bind to the modulatory domain of IP3R, elucidation of substances that bind to the modulatory domain of IP3R leads to elucidation of the function of IP3R, which will be very useful for therapy and diagnosis of a variety of diseases that are caused by the same.
Carbonic anhydrase-related protein (CARP) is a protein identified by screening genes specific for a Purkinje cell (a large nerve cell of cerebellar cortex, having dendrites arranged on the surface crossing a piriform cell body and flocculus) (see prior art document 8, Karo, K., (1990), FEBS Lett., 271, 137-40). CARP comprises 291 amino acids, and has an acidic amino acids cluster of 16 glutamic acid (Glu) residues and 4 aspartic acid (Asp) residues within the N-terminal 50 amino acids. CARP has a main carbonic anhydrase motif, but lacks carbonic anhydrase activity due to the absence of catalytic zinc coordinating residues. Indeed, it has been reported that CARP has no carbonic anhydrase activity. Also the human CARP gene has been cloned (see prior art document 9, Skaggs, L. A., Bergenhem, N. C., et al. , (1993), Gene, 126, 291-221), and shown to have 98% homology in amino acid residues to that of mouse, suggesting that CARP is highly conserved in the course of evolution. However, at the present stage, neither elucidation of function of CARP nor comprehensive elucidation about tissue distribution has been achieved, although it has been found that CARP is highly expressed in a Purkinje cell of cerebellum.
Prior art documents in relation to the invention of the present application are as follows.    1. Japanese Patent Application Laid-open No. 8-134099    2. Japanese Translation of PCT International Application No. 8-502068    3. Japanese Patent Application Laid-open No. 2000-135095    4. Furuichi, T., Yoshikawa, S., et al., (1989), Nature, 342, 32-38    5. Blondel, O., Takeda, J., et al., (1993), J. Biol. Chem., 268, 11356-11363    6. Yamamoto-Hino, M., Sugiyama, T., et al., (1994), Receptors Channels, 2, 9-22    7. Furuichi, T., Kohda, K., et al., (1994), Curr. Opin. Neurobiol., 4, 294-303    8. Karo, K., (1990), FEBS Lett., 271, 137-40    9. Skaggs, L. A., Bergenhem, N. C., et al., (1993), Gene, 126, 291-221    10. Bultynck G, De Smet P, et al., (2001), Biochem J., 354, 413-22